Notch1在肌成纤维细胞转化中的作用及表达变化

Function and expressive change of Notch1 during transformation of myofibroblasts

摘要:

目的  探讨在成纤维细胞转化为肌成纤维细胞过程中，Notch1表达的变化规律，以及γ-分泌酶抑制剂（DAPT）抑制Notch信号后，对细胞转化的影响。方法  取新出生2～3 d SD大鼠的肺组织，用胰酶消化法分离肺成纤维细胞，再将培养至第3代的细胞分为3组：对照组、转化生长因子-β1（TGF-β1）组、TGF-β1+
DAPT组。对照组为空白对照，TGF-β1组加入5ng/ml TGF-β1，TGF-β1+DAPT组同时加入5 ng/ml TGF-β1及5μmol/L DAPT。采用免疫细胞化学法对α-平滑肌肌动蛋白（α-SMA）的表达变化进行检测。同时通过逆转录聚合酶链反应（RT-PCR）及Western blot检测Notch1 mRNA和蛋白的表达变化。结果  α-
SMA免疫细胞化学结果表明，对照组大部分细胞无染色，而TGF-β1组则可见大部分细胞内有黄色和棕黄色颗粒及条纹，DAPT组和对照组无明显差异。对照组、TGF-β1组、TGF-β1+DAPT组Notch1 mRNA表达量分别为（0.278±0.022）、（0.783±0.018）和（0.313±0.029），对照组与TGF-β1组、TGF-β1组与TGF-β1+DAPT组比较，差异有统计学意义（P <0.05），对照组与TGF-β1+DAPT组比较差异无统计学意义（P >0.05）。对照组、TGF-β1组、TGF-β1+DAPT组Notch1蛋白表达量分别为（0.312±0.019）、（0.701±0.026）和（0.345±0.022），组间比较结果同Notch1 mRNA。结论  Notch1可以促进成纤维细胞向肌成纤维细胞的转化，进而促进肺纤维化。

Abstract:

Objective To study the law of changes of Notch1 expression in the transformative procession from fibroblasts to myofibroblasts and the effect of DAPT inhibition of Notch signal on cell transformation. Methods The lung tissue was taken from new-born 2 or 3 day Sprague-Dawley rats. Fibroblasts were separated with pancreatin digestion method. The third-generation cultivated cells were divided into three groups: comparison group, TGF-β1 group and TGF-β1+DAPT group. The comparison group was labeled as the control group, the TGF-β1 group was added with 5 ng/ml TGF-β1, and the TGF-β1+DAPT group was added with 5 ng/ml TGF-β1 and 5 μmol/L DAPT. Immunocytochemistry was used to assess change of α-smooth muscle actin (α-SMA) expression. RT-PCR was used to assess change of Notch1 mRNA expression. Western blot was used to assess change of Notch1 protein expression. Statistical software SPSS 18.0 was used for the data analysis with t-test. Results α-SMA immunocytochemistry demonstrated the majority of cells in the control group were not stained, the majority of cells in the TGF-β1 group had yellow and brown-yellow granules and stripes, and there was no obvious difference between the DAPT group and the control group. The Notch1 mRNA expressive value of the control group, the TGF-β1 group and the TGF-β1+DAPT group was (0.278 ± 0.022), (0.783 ± 0.018) and (0.313 ± 0.029) respectively; the Notch1 protein expressive value of the control group, the TGF-β1 group and the TGF-β1+DAPT group was (0.312 ± 0.019), (0.701 ± 0.026) and (0.345 ± 0.022) respectively; there were significant differences in the mRNA and protein levels of Notch1 between the TGF-β1 group and both the control and the TGF-β1+DAPT groups (P < 0.05), while the control group and the TGF-β1+DAPT group had no statistical differences (P > 0.05). Conclusions Notch1 can promote transformation of fibroblasts into myofibroblasts, and then promote pulmonary fibrosis.